采用超高效液相色谱-串联质谱法测定血浆中高蛋白结合率华法林的游离浓度及其与国际标准化比值的相关性。
Measurement of free concentrations of highly protein-bound warfarin in plasma by ultra performance liquid chromatography-tandem mass spectrometry and its correlation with the international normalized ratio.
作者信息
Huang Chenrong, Yang Jian, Du Yonghong, Miao Liyan
机构信息
Clinical Pharmacology Research Laboratory, First Affiliated Hospital of Soochow University, Suzhou 215006, China.
出版信息
Clin Chim Acta. 2008 Jul 17;393(2):85-9. doi: 10.1016/j.cca.2008.03.008. Epub 2008 Mar 21.
BACKGROUND
A novel method has been developed for the determination of free warfarin in human plasma by ultra performance liquid chromatography combined with tandem mass spectrometry (UPLC-MS/MS).
METHODS
The plasma filtrate was prepared by a high-speed ultrafiltration (UF), and was then extracted by a liquid/liquid extraction (LLE) with ethyl acetate. The chromatographic separation was performed on an Acquity UPLC BEH C(18) (2.1 mm x 50 mm, 1.7 microm, Waters) with a mobile phase consisting of 0.1% formic acid/acetonitrile (50:50, v/v). The analyses were carried out by multiple reaction monitoring (MRM) using the precursor-to-product combinations of m/z 307.3-->161.2 for warfarin and m/z 347.2-->161.3 for Cl-War.
RESULTS
The lower limit of quantification (LLOQ) was 0.25 ng/ml and the assay exhibited a linear range of 0.25-16 ng/ml and gave a correlation coefficient (r) of > or = 0.9999. Quality control samples (0.5, 2, 8 ng/ml) in 5 replicates from 3 different runs of analysis demonstrated intra-assay CVs of 3.0-10.2%, inter-assay CVs of 4.2-12.0%, and an overall accuracy of 85-115%.
CONCLUSIONS
The method can be applied to analyze the correlation of free concentration of warfarin and the international normalized ratio (INR).
背景
已开发出一种通过超高效液相色谱-串联质谱法（UPLC-MS/MS）测定人血浆中游离华法林的新方法。
方法
通过高速超滤（UF）制备血浆滤液，然后用乙酸乙酯进行液-液萃取（LLE）。在Acquity UPLC BEH C(18)（2.1 mm×50 mm，1.7 µm，沃特世）色谱柱上进行分离，流动相为0.1%甲酸/乙腈（50:50，v/v）。采用多反应监测（MRM）模式进行分析，华法林的母离子-子离子对为m/z 307.3→161.2，氯华法林的母离子-子离子对为m/z 347.2→161.3。
结果
定量下限（LLOQ）为0.25 ng/ml，该方法的线性范围为0.25 - 16 ng/ml，相关系数（r）≥0.9999。来自3次不同分析运行的5个重复的质量控制样品（0.5、2、8 ng/ml）的批内变异系数为3.0 - 10.2%，批间变异系数为4.2 - 12.0%，总体准确度为85 - 115%。
结论
该方法可用于分析华法林游离浓度与国际标准化比值（INR）之间的相关性。
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